Regulation of long-term oviposition in the house cricket Acheta domesticusRoles of
prostaglandin and factors associated with sperm.

код для вставки на сайт или в блог

ссылки на документ

Archives of insect Biochemistry and Physiology 659-72 (1987)
Regulation of Long-Term Oviposition in the
House Cricket, Acheta domesticus: Roles
of Prostaglandin and Factors Associated
With Sperm
Michael P. Murtaugh and David L. Denlinger
Department of Entomology, Ohio State University, Columbus
The transfer of spermatophore contents derived from testes during mating
greatly stimulates ovipositional activity for long periods of time in the house
cricket, Acheta domesticus (L.). Since prostaglandins appear t o play a role in
reproduction i n several insect species, and since prostaglandin synthesis
enzymes occur in cricket testes and spermatophores, we investigated the
role of prostaglandins i n the regulation of long-term oviposition. Inactivation
of prostaglandin synthesis enzymes in males or females using specific
inhibitors failed to block mating-induced increases in egg laying. However,
males lacking sperm because of X-irradiation were unable t o induce
oviposition even though they mated, transferred spermatophores, and had
high levels of prostaglandins in both testes and spermatophores. X-irradiation
was also used t o generate males with nonfunctional sperm. Females mated
t o these animals readily laid eggs, which failed t o develop. It appeared that
sperm or a factor associated with sperm induced long-term oviposition in
female house crickets. Prostaglandin synthesis enzymes transferred from the
male t o females may have other roles in the female, for example, in sperm
maintenance in the spermatheca. Previous observations strongly suggest that
prostaglandins induce egg laying behavior and activity; they may be
synthesized by female enzymes that are regulated by male-derived factors.
Key words: X-irradiation, PGE, PGF2,
INTRODUCTION
In many insects, oviposition is induced or stimulated by male factors that
are transferred during mating and act in females El]. Ovipositional stimuli
Acknowledgments: We thank Laura Wiebers for preparing the manuscript. This research was
supported by a Graduate Fellowship from the Ohio State University Graduate School to
M.P.M., by a Sigma Xi Grant-In-Aid-of-Researchto M.P.M., and by grant 8600186 from the
USDA Competitive Research Grants Office.
Received Janary2,1987; accepted May 8,1987.
Michael Murtaugh is now at the Department of Veterinary Pathobiology, University of Minnesota, 1971 Commonwealth Avenue, St. Paul, M N 55108. Address reprint requests to this
address.
0 1987 Alan R. Liss,
inc.
60
Murtaugh and Denlinger
range from mechanical, the mating act itself, to chemical inducers secreted
by various male reproductive organs [reviewed in 11. The transfer of malederived ovipositional stimuli with sperm at the time of mating serves to
coordinate fertilization with oviposition and thus increases reproductive
success.
In the house cricket, Acketa domesticus (L.), mating is a powerful stimulus,
which promotes oviposition at a high rate for many weeks 121. The malederived house cricket ovipositional stimulus is of some interest in that it
appears to be both potent and stable. A single mating early in adult life
stimulates the laying of fertilized eggs for 60 days, and the inducing activity
is continuously present throughout this period [2]. In addition, it can persist
in recently emerged females, which mate readily but do not lay eggs for 1014 days.
The biochemical nature of the male-derived ovipositional stimulus has not
been determined, but enzymes and products of the prostaglandin (PG)
synthesis system are present in male reproductive tissues and have been
implicated in inducing oviposition [2-71. Prostaglandins are unsaturated,
long-chain fatty acids that are found in all orders of animals and have diverse
biological activities. PGs are widely distributed in insects in many tissues
and organs. Their physiological function in insects has been studied most
intensively with respect to reproduction, in which the E-type PGs appear to
be involved in the regulation of the egg laying process. In crickets, the
elevation of PGE in female reproductive tissues causes a short-lived ovipositional response [6,8,9]. However, whether prostaglandins also stimulate the
long-term ovipositional activity of house cricket females has not been
resolved.
In this paper, we examine the role of PGs in the regulation of long-term
oviposition in house crickets. The results obtained from these studies suggest
that PGs do not mediate the long-term egg laying response in the house
cricket. Rather, a factor associated with sperm appears to be the principal
male-derived ovipositional stimulus in these insects.
MATERIALS AND METHODS
Insect Maintenance and Egg Collection
A. domesticus (L.) adults and mid- to late-instar nymphs were maintained
in continuous culture as described previously [3]. Oviposition was monitored
by holding mated females in 1pint containers with screen bottoms on moist
perlite (Terra-lite, W.R. Grace, Cambridge, MA) as described previously [3].
PG Measurement
PGE and PGF2, levels in cricket tissues were determined by radioimmunoassay as described elsewhere
Data shown were adjusted for losses
during purification [7]. The extraction efficiency, which varied with the
amount of starting tissue, averaged 35-40%
[n.
[n.
Pharmacological Inhibition of PG Synthesis
Aspirin, acetaminophen, and indomethacin from Sigma (St. Louis, MO)
were administered by addition to the food in a concentration of 1%or, for
Sperm Regulates Cricket long-Term Oviposition
61
aspirin, 5%. Acetaminophen was also provided as a saturated solution in
water and was available ad libitum.
X-Irradiation
Adult males age 1-4 days were exposed to 250-8,000 rads of X-irradiation
delivered from a Norelco MG X-ray tube operated at 250 KV and 10 mA.
Cricket juveniles of mixed sex, and approximately at the stage when external
genitalia appear, were given 1,200 rads from the X-ray tube operated 150 KV
and 10 mA.
RESULTS
Inhibition of PG Synthesis in Males
Previous studies showed that aspirin and acetaminophen, which were
available ad libitum in the food and water, respectively, for a period of 10-20
days, decreased PGE levels in the testis by 91% and PGF2, levels by 64% [fl.
Indomethacin at a concentration of 1%in food completely inhibited PG
synthesis [7l. Inhibition of testicular PG synthesis also was reflected in
reduced PG levels in spermatophores. The level of PGE (15 rt 4 pglspermatophore, n=25) was decreased 70% by aspirin and acetaminophen in these
experiments. PGF2, was not detected in spermatophores from control or
treated animals. Thus, continuous administration of PG synthesis inhibitors
to adult males profoundly suppressed testicular PG levels.
Inhibition of PG Synthesis in Males: Effect on Induction of Oviposition
Figure 1 shows the ovipositional activity of females after mating with
males that had been administered inhibitors of PG synthesis. The inhibition
of enzymes involved in PG synthesis in male reproductive tissues and the
reduction of PG levels in spermatophores failed to affect the pattern of egg
laying by females mated to them. In females mated to both treated (Fig. 1,
closed circles) and untreated (Fig. 1, open circles) males, oviposition was
greatly stimulated within 4 days and continued at a high rate until the
experiment was terminated after 12 days.
The males referred to above were treated with aspirin and acetaminophen
for 12-13 days, beginning on the day after adult ecdysis. However, in crickets, sperm development occurs prior to adulthood, so, at the onset of adulthood, the testis contains spermatozoa at all stages of differentiation [lo].
Thus, if PGs were required during sperm development or in the early stages
of spermatogenesis, then inhibition of PG synthesis during adulthood would
not affect the large number of spermatozoa that had already differentiated.
Hence, a group of young nymphs that had not yet elaborated external
genitalia were placed on the inhibitor diet. As adults, these males, which
were 23-40 days old and had been on inhibitor diet as juveniles and adults
for 61 days at the time of mating, induced oviposition just as proficiently as
normal males (Fig. 1, open and closed squares). Furthermore, sperm viability
as measured by egg hatchability indicated that sperm from males maintained
on PG synthesis inhibitors was equivalent to sperm from untreated males.
62
Murtaugh and Denlinger
Days a f t e r mating
Fig. 1. Effect of PG synthesis inhibitors in the male on egg laying activity. One-day-old males
were provided with untreated food and water (0)or food with 5% aspirin and water
saturated with acetaminophen ( 0 ) After
.
12-14 days, each male mated with one female. Eggs
were collected for 12 days after mating. Data represent the mean number of eggs laid per day
per female for five females mated to untreated males and for 13 females mated to treated
males. In a second experiment, eggs were collected from females mated at 16-18 days of
adult age t o individual males. Males either were untreated, 16-day-oldadults (El)or were 2340-day-old adults reared for a total of 61 days on food and water containing aspirin and
acetaminophen (M)as described. Data represent the mean number of eggs laid per day by
three females (0)
or 18 females (M).Standard errors averaged 35% of the mean in the
untreated group and 20% of the mean for the treated group.
Eggs laid by females mated to untreated males hatched at a rate of &-82%
(910 eggs examined) over the 10 day oviposition period. The fertility of eggs
laid by females mated to treated males varied from 77% to 90% (5,904 eggs
examined). The results of the preceding studies indicated that pharmacologic
inhibition of PG synthesis enzymes in males before mating did not affect the
pattern or the frequency of oviposition in mated females, nor did it affect
embryogenesis and hatching in deposited eggs.
Inhibition of PG Synthesis in Females
PG levels are increased greatly in the female spermatheca after mating
[6,7,11]. For example, PGE, which was not detected in virgin spermathecae,
averaged 215 pglgland in mated animals [ 7 ] . The administration of aspirin
and acetaminophen to females completely blocked the mating-induced increase in PG levels [7]. An equally effective inhibition of PG synthesis in the
spermatheca after mating was also observed when the inhibitors were fed to
males [ 7 ] .
The effect on egg deposition of PG inhibitor administration to females is
shown in Figure 2. Untreated females displayed a burst of ovipositional
activity immediately after mating followed by a lower but sustained level of
activity for 8 days (Fig. 2, closed circles). Females fed aspirin and acetamino-
Sperm Regulates Cricket Long-Term Oviposition
63
Days a f t e r m a t i n g
Fig. 2. Effect of administering PG synthesis inhibitors to females on subsequent egg laying.
Females were placed on diets containing aspirin and acetaminophen from the day of eclosion
until they were mated 16-17 days later to untreated males. Eggs were collected at 2 day
intervals. Data shown are the means and standard errors from 18 untreated females ( 0 )and
12 treated females (0).
phen for 16 days before mating showed a low rate of egg deposition which
increased to the control rate in 7-8 days (Fig. 2, open circles). A similar
stimulation of long-term egg laying activity without an initial burst was
observed in two additional experiments (not shown). The lack of a large
burst of egg laying activity immediately after mating in these experiments
was attributed to nonspecific effects of aspirin and acetaminophen that were
unrelated to PG synthesis in reproductive tissues, since, as indicated previously, postmating increases in spermathecal PG levels were blocked totally
by treating males with PG inhibitors
a procedure that had no effect on
ovipositional activity (see Fig. 1).
PG synthesis inhibitors in females have no apparent effect on the survivability of deposited eggs. The frequency of hatching in three experiments in
which females received maximally effective levels of aspirin and acetaminophen throughout the period of oogenesis varied from 76% to 80% in a total
of 1,361 eggs examined.
[n,
PGs and Egg Deposition in Young Females
The preceding experiments attempted to evaluate the role of PGs in
stimulating oviposition by observing the effects of PG synthesis inhibitors in
mature females that were competent to lay eggs as determined previously
[3]. The results did not indicate that PGs mediate or constitute the matinginduced stimulation of egg laying. However, another feature of house cricket
reproductive physiology is that young adult females mate readily but do not
Murtaugh and Denlinger
64
lay eggs until approximately days 12-14 of adulthood [3]. To determine if this
delay in oviposition was due to the failure of mating to increase spermathecal
PG levels, PG levels were measured in the spermatheca of young mated
females. Four- to six-day-old females were mated, and spermathecae were
removed 24 h later for determination of PGE and PGF2, levels. These individuals would not be expected to commence oviposition until about 7 days later.
Nevertheless, high levels of spermathecal PGE were detected (Table 1).
Furthermore, these values were substantially higher than in mature females
treated with PG synthesis inhibitors [7], which were able to lay eggs (see Fig.
2).
We also attempted to overcome the ovipositional delay in young females
with exogenous PG. Four- to five-day old females were injected with PGE2
or were mated. Comparison of the egg laying behaviors of injected and
mated females is shown in Figure 3. A pharmacological dose (100 pg) of
PGE2 was completely ineffective, failing to elicit any egg laying in young
females (Fig. 3, closed circles). Females from the same group that were
allowed to mate commenced prolonged ovipositional activity 5 days later
(Fig. 3, open circles). Injection of 4-5 day-old-females with PGF2, (100 pg)
likewise failed to elicit ovipositional activity (data not shown).
Injection of PGE2 into 20-day-old adult females induced a 1 day burst of
egg laying similar to the short-term effects of PGE2 injection reported previously [6,8,9]. One hundred micrograms of PGE2 administered intraabdominally induced females to lay an average of l2+1 eggs in the following 24 h
as compared to lrt0.2 eggs per female in buffer-injected controls (sample
size = 15-8 crickets per group). PGF2, at 100 &female was not significantly
different from controls. In the following six days, all groups laid less than
one egg per female per day. Identical results were obtained irrespective of
incubation temperatures from 25 to 31°C.
Effect of X-Irradiation on Male Stimulation of Egg Laying
From the previous experiments, it appeared that PGs or active PG synthesis enzymes from the male were not required to stimulate long-term egg
laying in females. Furthermore, the accumulation of PGs in female reproductive tissue was not sufficient to induce ovipositional activity. Therefore, we
evaluated other components of the spermatophore for egg laying stimulatory
activity. A likely candidate was sperm, since the use of sperm to induce egg
TABLE 1. Effect of Prostaglandin Synthesis Inhibitors on Prostaglandin Levels in the
Spermathecae of Young Females*
Experiment
No. of
individuals
1
2
3
12
13
13
PGE (pg) per
Individual
mg Protein
90
90
54
550
464
320
PGF2, (pg) per
Individual
mg Protein
18
46
36
107
239
214
*Four- to six-day-old females were mated to untreated males. After mating, spermathecae
were removed for PG determinations. Protein determinations were carried out according to
Bradford [25] after chloroform extraction of the samples to remove lipids.
Sperm Regulates Cricket long-Term Oviposition
65
Days after t r e a t ment
Fig. 3. Effect of PGE2 or mating on house cricket oviposition. Four- to five-day old adult
females were mated (0,n = 18) or were injected with 100 p g of PGE2in 5 pI of 20 mM sodium
phosphate, pH 7.4, through an intersegmental membrane of the ventral abdomen ( 0 ,n =
20). Data represent the mean and standard error. Results over the first four days were the
same for both groups.
laying would be the simplest method to link oviposition with fertilization. To
examine the role of sperm or factors associated with sperm in the matinginduced stimulation of egg laying, X-irradiation was used to deplete males of
sperm. X-irradiation damages chromosomal DNA and interferes with meiosis
and mitosis. Thus developing sperm fail to differentiate, and mature spermatozoa are incapable of fertilization.
In these experiments, it was important that treatment with X-irradiation
specifically block spermatogenesis without affecting PG biosynthesis. To
determine whether X-irradiation affected PG synthesis in reproductive tissues, males were irradiated as nymphs and PG levels were determined in
the testes and spermatophores of adults at various intervals. The intensity of
irradiation, 1,200 rads, was sufficient to block sperm development completely, as shown below but development of somatic tissue was unaffected.
Treated animals molted successfully and survived as adults at the same rate
as untreated siblings. The levels of PGE and PGF2, in testes of irradiated
males were striking (Fig. 4).At the earliest time tested, PGE and PGF2, levels
were greater than those in untreated controls. At later times, levels of both
PGs were even higher. The data shown in Figure 4 are expresssed on a per
individual basis. Treated testes lack sperm and were significantly smaller, as
indicated by protein determinations (Fig. 4, inset). Thus the PG concentrations were even greater on a mass basis. PGE levels in spermatophores of
irradiated males were about the same (10-30 pglspermatophore) as in unirradiated males. PGF2,, which was not detected in spermatophores of normal
males, was abundant in irradiated males (10-100 pglspermatophore). The
high concentration of PGs observed in both testes and spermatophores of
66
.
Murtaugh and Denlinger
2400-
br
0
2000 ((I
3
'P 1600>
10
I4
C
(3
a.
21
18
16
adult age
'0
1200-
a
800 -
400 -
0-
-
10
0
Y
14
18
22
26
Adult age at analysis
Fig. 4. Effect of X-irradiation on PG levels in testes. Males were irradiated as nymphs 30-41
days before PG measurement. Each data point i s based on an assay of eight to 18 individuals.
PGE (M) and PGF, (0)
levels in testes of untreated 23-day-old males are shown for comparison. Inset: protein content of the testes in irradiated ( @ ) and untreated (0)males. The
samples used for protein determinations were taken from the pooled samples used for PC
measurements.
irradiated males indicated that the enzymes involved in PC biosynthesis
were present and were active.
Disruption of normal chromosome function and sperm inactivation was
assessed by the inability of sperm from irradiated adult males to fertilize
eggs. The data in Figure 5 show that increasing doses of X-rays up to 4,000
rads caused infertility in males. Figure 5 also shows that the process of
irradiation itself had no effect on the ability of males to induce oviposition
(closed circles). Infertility was not due to the absence of sperm; inspection of
spermathecae from females at the end of the experiment showed that in
every case sperm were present.
The tissue damage induced by X-irradiation was specific for the reproductive system in males and females. Treatment of juveniles with 1,200 rads Xirradiation completely blocked reproductive development, but treated animals continued to develop and molt. They reached adulthood as rapidly as
untreated siblings, and their adult longevity was uninpaired. Yet, if irradiated
10 days before adult eclosion, the development of reproductive tissue was
severely retarded. In irradiated females, 12-13 days after the final molt, the
median ovarian wet weight was 4.5 mg (n=10) and no eggs were found. In
untreated siblings, median weight was 156 mg (n=10), and ovaries were
filled with eggs. In males, irradiation of juveniles caused lack of sperm
production as adults and testes were reduced in size. However, as adults,
Sperm Regulates Cricket long-Term Oviposition
67
I
Y
v)
x
m
-0
u)
(J,
(J,
W
Fig. 5. Effect of X-irradiation of males on oviposition and fertility in females to which they
were mated. Males were irradiated as described in Materials and Methods at 1-4 days of
adult age. After 7 days, males were allowed to mate with IS-23-day-old females. Each point
represents t h e mean number of eggs laid per female in 8 days ( 0 )or percent egg fertility
(0)
for 6-8 females.
these animals manufactured spermatophores and transferred them successfully to females.
Irradiation at earlier development times, before spermatozoa were present
in the testes, blocked sperm development. Thus irradiation effectively froze
sperm development so that no further differentiation would take place.
Males irradiated 11-12 days before eclosion with 1,200 rads produced spermatophores with sperm as 10-11-day-old adults, but testicular growth was
greatly retarded: Median wet weight of these testes was 18.5 mg (n=lO),
whereas that in a group of siblings reared under identical conditions was
43.5 mg (n=10). Increasing the dosage of X-rays administered to penultimateinstar nymphs increased the percentage of mature males lacking sperm.
When these males mated with females, the effects on oviposition depended
dramatically on whether sperm were present (Fig. 6). At 400 rads, 72% of
surviving males transferred sperm to females. In each case, long-term oviposition was stimulated. At 800 rads, only 18% of males transferred sperm,
but again in these cases oviposition was stimulated. At 1,600 rads, no males
transferred sperm. At every dosage level, males that failed to transfer sperm
failed to elicit an ovipositional response even though spermatophores were
attached, as determined in all cases by visual observation.
Treating juvenile crickets at midinstar ages with X-irradiation produced
mature males whose spermatophores were usually devoid of sperm. Following mating with these males, females failed to oviposit (Fig. 7, cross-hatched
bars). Occasionally, sperm were transferred (determined by examination of
the spermathecal contents at the end of the experiment), and in these cases
68
Murtaugh and Denlinger
sperm t r m r f e r r e d
El sperm
not transferred
n:5
L
400
800
ii
1600
X-ray dose to male (rads)
Fig. 6 . Effect of various doses of X-rays on sperm transfer and oviposition in the house
cricket. Last-instar male nymphs were irradiated, and 41 days later (as 30-38-day-old adults)
surviving males were each given two to five females. After mating, eggs were collected for 7
days. Data represent the mean number of eggs laid per day per female. The numbers
displayed above each bar are the number of mated females that did (stippled bars) or did not
(open bars) receive sperm. No males given 1,600 rads of X-rays transferred sperm at mating.
egg laying was induced (Fig. 7, open bars). However, in these animals, the
rate of oviposition declined steadily during the 10 day period, in contrast to
that of females mated to untreated males (Fig. 7, solid bars).
It appeared from these results that an intact PG synthesis system was not
a sufficient stimulus to provoke oviposition in mated females but that the
presence of sperm, even nonviable sperm, was required to induce oviposition. The last experiment was to determine if the presence of sperm was
necessary to stimulate the delayed oviposition response of young females.
Therefore, 2-4-day-old females were mated to X-irradiated or untreated males
and provided with an oviposition substrate for 20 days. Females that received
sperm from normal or irradiated males (Fig. 8) exhibited an elevated rate of
egg deposition 8 days after mating, which persisted to the end of the experiment. The data for those mating to untreated or irradiated males was combined since the numbers were indistinguishable. Mated females that failed
to receive sperm as determined by examination of spermathecal contents laid
eggs at the same rate as unrnated control crickets (Fig. 8, open circles and
open squares).
DISCUSSION
In crickets, especially A. domesticus and Teleogryllus cornrnodus, female egg
laying is greatly stimulated by spermatophore contents derived from the
Sperm Regulates Cricket long-Term Oviposition
69
100-
2 80m
-0
0
2
.
5
.
c
60
a,
m
40
*-
v)
u,
u,
w
20
2
4
8
6
Days after
10
mating
Fig. 7. Effect of X-irradiation on sperm transfer and oviposition in house crickets. Males 1316 days old received 1,200 rads of X-irradiation 48 days before mating to individual females.
Of 25 females that accepted spermatophores, four had sperm in the spermatheca (open bars)
and 21 did not (cross-hatched bars). Five females mated to untreated siblings had sperm
(solid bars). Data represent means and standard errors.
4
8
1:
Days after mating
Fig. 8. Oviposition by female house crickets after mating at an early age with X-irradiated
males. Two- to 4-day-old females were mated to X-irradiated or untreated males 10-13 days
old or were not mated. Closed circles represent the means of seven mated females that
received sperm from five normal and from two X-irradiated males. Open circles represent
means of 22 mated females that did not receive sperm. Open squares represent means of
seven unmated females.
70
Murtaugh and Denlinger
male testis [12,13]. This observation, combined with the finding of PGs and
PG synthesis enzymes in testes and spermatophores [4,5,7,11], has led to the
suggestion that PGE2, or the enzymes responsible for its synthesis, are the
male-derived oviposition stimulus. Additional experiments, including the
inhibition of egg deposition in crickets by inhibitors of PG synthesis [5], and
the induction of egg laying in crickets by the injection of PGE2 [6],supported
this hypothesis. However, the episodes of egg laying involved in these
experiments were of short duration, typically lasting about 24 h. We were
interested in determining if the same stimulus was responsible for inducing
the prolonged period of egg deposition lasting for a period of weeks that
commences after A. domesticus females mate [3].
PG synthesis inhibitors were used to evaluate the role of PGE2 and PGF2,
in the induction of long-term oviposition. Aspirin and acetaminophen irreversibly inhibit PG endoperoxide synthase [14-17'J and prevent large postmating increases in PGE and PGF2, levels in the spermatheca [q.
Administration of these inhibitors to males blocked PG synthesis in the testis
[7] and, presumably, results in the packaging of inactive synthetase in spermatophores. Nevertheless, these inhibitors, when given to males, do not
block mating-induced oviposition. We were unable to detect any effect of
inhibitors on oviposition when they were administered to males. Both young
females and older, ovipositionally competent, females displayed normal egg
laying patterns after mating with inhibitor-treated males. Thus a contribution
of PGs or active PG synthesis enzymes from the male was not required to
stimulate oviposition.
In females, PG synthesis inhibitors acted in a more complex manner. Egg
laying by mated females initially was suppressed in animals given diet
containing inhibitors, but, after several days without inhibitor egg laying
activity began. From these results, it appears that PG synthesis in the female
is important in the ovipositional process. Combined with the results from
inhibiting PG synthesis in the male, these observations indicate that femalederived PGs play a physiological role in the egg laying process. If PGs are
required for the ovipositional event itself, then these experiments cannot
address questions about earlier events associated with induction of long-term
ovipositional responses. If PG synthesis in the female reproductive tract is
required for induction of the long-term response, then these experiments
indicate that the male-derived factor functions by activating or inducing
female PG synthesis enzymes. These data are interesting in light of previous
observations that an intact spermatheca with intact neural connections is
required for egg laying [3].Thus oviposition at some point involves interaction with the nervous system. It is also clear from the experiments with
young females that the induction of egg laying involves much more than
increasing PG levels in female reproductive tissues. These animals have
mature eggs 131, are mated, and have high levels of PG (Table 1)yet will not
lay eggs for another 5-7 days [ 3 ] .
In a variety of insect species, sperm have been implicated as an inducer of
egg laying [18-221. Our studies indicate that sperm or factors associated with
sperm induce egg laying in the house cricket. In contrast to the lack of effect
of PG synthesis inhibitors on male-induced egg laying, elimination of sperm
Sperm Regulates Cricket Long-Term Oviposition
71
from testes and spermatophores rendered males incapable of stimulating
oviposition. Removal of sperm from the insemination mixture was accomplished by irradiating adult or immature males with X-rays. The effect of Xirradiation to abolish the egg laying stimulus was due specifically to the
removal of sperm, since the application of even very high doses of X-rays to
males did not interfere with the induction of oviposition if sperm were
transferred during mating (Fig. 5). Furthermore, treatment of males with Xrays did not impair PG synthesis. In fact, it appeared to enhance PG synthesis; irradiated males had extremely high levels of both PGE and PGF2, in
testicular tissue and in spermatophores. Ionizing radiation has been associated with increased PG synthesis [23], an effect that may be due to the
inhibition of thromboxane A2 biosynthesis with resultant overproduction of
other PGs 1241. Thus, in irradiated males, enzyme complexes involved in PG
biosynthesis were active.
Previous studies showed that mating provided a continuous stimulus for
oviposition which could be titered out by removing the spermatophore at
short time periods after transfer [3]. The data in Figure 6, showing that egg
laying activity and sperm production diminished as X-ray dosage increased,
also indicate that the presence of sperm is required for egg laying activity.
Likewise, the transfer of small numbers of sperm would be expected to
stimulate a burst of ovipositional activity that would decline as sperm were
depleted, as is shown in Figure 7.
The results of these experiments strongly suggest that sperm contain the
information that induces prolonged ovipositional activity in mated females.
PGs appear to have a role in episodic egg laying and perhaps in other, not
yet defined, aspects of reproduction.
LITERATURE CITED
1. Englemann F: The Physiology of Insect Reproduction. Pergamon Press, Oxford (1970).
2. Brady UE: Prostaglandins in insects. Insect Biochem 13, 443 (1983).
3. Murtaugh MP, Denlinger DL: Physiological regulation of long-term oviposition in the
house cricket, Achetu domesticus. J Insect Physiol31, 611 (1985).
4. Destephano DB, Brady UE, Lovins RE: Synthesis of prostaglandin by reproductive tissue
of the male house cricket, Achetu domesticus. Prostaglandins 6, 71 (1974).
5. Destephano DB, Brady UE: Prostaglandin and prostaglandin synthetase in the cricket,
Achetu domesticus. J Insect Physiol23, 905 (1977).
6. Loher W, Ganjian I, Kubo; I, Stanley-Samuelson D, Tobe SS: Prostaglandins: Their role
in egg-laying of the cricket TeleogryZlus commudus. Proc Natl Acad Sci USA 78, 7835 (1981).
7. Murtaugh MP, Denlinger DL: Prostaglandins E and F2a in the house cricket and other
insects. Insect Biochem 12, 599 (1982).
8. Loher W: The influence of prostaglandin E2 on oviposition in Teleogvyllus cornmodus.
Entomol Exp Appl25, 107 (1979).
9. Destephano DB, Brady UE, Farr CA: Factors influencing oviposition behavior in the
cricket, Acheta domesticus. Ann Entomol SOCAm 75, 111(1982).
10. McMaster-Kaye R, Kaye JS: Basic protein changes during the final stages of sperm maturation in the house cricket. Exp Cell Res 97, 378 (1976).
11. Stanley-Samuelson DW, Klocke JA, Kubo I, Loher W: Prostaglandins and arachidonic acid
in nervous and reproductive tissue from virgin and mated female crickets, TeZeogyllus
cornmodus. Entomol Exp Appl34, 35 (1983).
72
Murtaugh and Denlinger
12. Murtaugh MP, Kapoor CL, Denlinger DL: Extracellular localization of cyclic GMP in the
house cricket male accessory reproductive gland and its fate in mating. J Exp Zoo1 233,413
(1985).
13. Loher W, Edson K: The effect of mating on egg production and release in the cricket
Teleogyllus cornrnodus. Entomol Exp Appl 16, 483 (1973).
14. Smith WL, Lands WEM: Stimulation and blockade of prostaglandin biosynthesis. J Biol
Chem 246: 6700 (1971).
15. Raz A, Stern H, Kenig-Wakshal R: Indomethacin and aspirin inhibition of prostaglandin
E2 synthesis by sheep seminal vesicles microsome powder and seminal vesicle slices.
Prostaglandins 3, 337 (1973).
16. Abdel-Halim MS, Sjoquist B, Anggard E: Inhibition of prostaglandin synthesis in the
brain. Acta Pharmacol Toxic01 43,266 (1978).
17. Demers LM, Budin RE, Shaikh BS: The effects of aspirin on megakaryocyte prostaglandin
production. Proc SOCExp Biol Med 163, 24 (1980).
18. Davey KG: Copulation and egg-production in Rhodnius prolixus: The role of the spermathecae. J Exp Biol42:373 (1965).
19. Davis NT: Studies on the reproductive physiology of Cimicidae (Hemiptera)-11. Artificial
insemination and the function of the seminal fluid. J Insect Physiol 11, 355 (1965).
20. Benz G: Influence of mating, insemination, and other factors on oogenesis and oviposition
in the moth Zeimphera diniana. J Insect Physioll5,55 (1969).
21. Truman JW, Riddiford LM: Role of the corpora cardiaca in the behavior of Saturniid
moths. 11. Oviposition. Biol Bull 140, 8 (1971).
22. Thibout E: Stimulation of reproductive activity of females of Acrolepiopsis assecteltu (Lepidoptera: Hyponomeutoidea) by the presence of eupyrene spermatozoa in the spermatheca. Entomol Exp Appl26, 279 (1979).
23. Mennie AT, Dalley VM, Dinneen LC, Collier HOJ: Treatment of radiation-induced gastrointestinal distress with acetyl salicylate. Lancet 2, 942 (1975).
24. Horrobin DF, Mariku MS, Karmali RA, Oka M, Ally AI, Morgan RO, Karmazyn M,
Cunnane SC: Thromboxane A2: A key regulator of prostaglandin biosynthesis and of
interactions between prostaglandins, calcium and cyclic nucleotides. Med Hypoth 4, 178
(1978)
25, Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities
of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248 (1976).
'